A vaccine to prevent infection with human immunodeficiency virus type 1 (HIV-1) should induce potent, broadly neutralizing antibodies(NA). Our preliminary studies of neutralization escape mutants from the MN strain have demonstrated the mutants to be broadly resistant to NA by an as yet undescribed mechanism, and have focused on the relative paucity of information on the mechanisms of escape mutation that may occur in vivo. We propose to further define mechanisms of resistance of HIV-1 to neutralization by the study of neutralization escape mutants that developed in vivo and in vitro. We are in the process of developing and establishing procedures that will be used in this effort. Envelope genes of interest will be copied by polymerase chain reaction(PCR) and cloned into an expression vector. These expression vectors will be used to express envelope antigen in COS7 cells and will be cotransfected into COS7 cells with HIV-gpt to produce pseudovirions capable of a single cycle of infection. Pseudovirions or envelope proteins that appear to represent escape mutants will be evaluated for antigenic reactivity with monoclonal antibodies and human sera, and for cell tropism, CD4 binding, and gpl20- gp4l association properties. We will use these and other methods to address each of the specific aims of this project The genetic basis for escape mutation of the MN strain variants we have developed will be determined, and biological characteristics associated with neutralization resistance of the variants will be studied. We will establish and study additional neutralization escape mutants in vitro from infectious molecular clones of six T cell line tropic and two macrophage tropic strains of HIV-1, including MN, NL4-3, SF2, Z2Z6, NY5, ALA1, NL(SF162), and AD8. The escape mutants will be selected by propagating the viruses at limiting dilutions in the presence of high titered neutralizing human sera. We will also study mutants developed in vivo which will be obtained from participants in the Multicenter AIDS Cohort Study(MACS) and from a cohort that has been studied extensively in our laboratory. Envelope genes in peripheral blood lymphocytes from these individuals will be cloned and studied. Immunological evaluations of escape mutants will include use of sera from human vaccinees, infected non-human primates, and immunized mice. The results of these studies should elucidate the potential mechanisms of antigenic variation of HIV- l, the relationships of these antigenic changes to other biological properties of the envelope glycoproteins, the potential for induction of broadly cross reactive NA by vaccination, and potential reasons for neutralization resistance of primary isolates of HIV-1.